The concept that cancer stem cells exist, and are able to regenerate themselves and all other cells in the malignant clone implies that any treatment not effective against such cells would ultimately fail when the residual stem cells began to proliferate and re-establish the tumor. The side population (SP) of normal bone marrow cells, which has a primitive CD34-low/negative phenotype, is a cancer stem cell candidate within the hematopoietic system. We have identified tumor SP cells in acute myeloid leukemia and, more recently, in B-cell chronic lymphocytic leukemia (B-CLL), and have shown that they possess features compatible with a cancer stem cell. These observations stimulated the central hypothesis of our proposal - that phenotypic differences between the SP and non-SP cells in B-CLL could be exploited to identify additional features of SP cells that might be useful targets for therapeutic intervention. We propose three specific research aims to pursue this goal. AIM 1: Because SP cells will not be clinically valuable unless they act as true cancer stem cells, we propose to target them in B-CLL patients by administering an autologous CD40L- and IL-2- expressing B-CLL vaccine over a prolonged (12-month) period in a Phase I trial. Progressive (but delayed) reduction of the non-SP tumor cells, as seen in our earlier study, would validate the stem cell candidacy of the malignant SP cells. AIM 2: to identify fundamental differences between tumor SP and non-SP cells, we will analyze and compare the expression of stem cell-related genes known to regulate quiescence, division and self-renewal in these two cell populations. AIM 3: to determine the target antigen(s) on B-CLL SP cells recognized by tumor-reactive immune cells generated during our tumor vaccine studies. Information from this project will (i) demonstrate the feasibility of enhancing immune responses against B-CLL and its SP cells by extending vaccinations already found to elicit responses against SP cells in B-CLL and (ii)reveal additional markers that distinguish SPfrom non-SP tumor cells, which could provide novel targets for immune-based or other forms of treatment. Establishing that tumor SP cells in B-CLL are truly crucial to sustaining the malignancy would have considerable implications for the treatment of other malignant diseases in which cancer stem cells may be present. Lav summary: Many cancers contain stem cells that resist standard therapy and regenerate the disease, even when all other tumor cells have been destroyed. This project will identify the cancer stem cells in a common adult leukemia, chronic lymphocytic leukemia, and then test immune system-based treatments that could eliminate the stem cells.